Detection of 100% of mutations in 124 individuals using a standard UV/Vis microplate reader: a novel concept for mutation scanning

We report the development of a simple and inexpensive assay for the detection of DNA polymorphisms and mutations that is based on the modification of mismatched bases by potassium permanganate. Unlike the chemical cleavage of mismatch assay, which also exploits the reactivity of potassium permanganate to detect genomic variants, the assay we describe here does not require a cleavage manipulation and therefore does not require expensive or toxic chemicals or a separation step, as mismatches are detected using direct optical methods in a microplate format. Studies with individual deoxynucleotides demonstrated that the reactivity with potassium permanganate resulted in a specific colour change. Furthermore, studies with synthetic oligonucleotide heteroduplexes demonstrated that this colour change phenomenon could be applied to detect mismatched bases spectrophotometrically. A collection of plasmids carrying single point mutations in the mouse β-globin promoter region was used as a model system to develop a functional mutation detection assay. Finally, the assay was validated as 100% effective in detecting mismatches in a blinded manner using DNA from patients previously screened for mutations using established techniques, such as sequencing, SSCP and denaturing high-performance liquid chromatography (DHPLC) analysis in DNA fragments up to 300 bp in length

Molecular Basis of Phenylketonuria in Italy

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A(1)ATVar: A Relational Database of Human SERPINA1 Gene Variants Leading to alpha(1)-Antitrypsin Deficiency and Application of the VariVis Software

We have developed a relational database of human SERPINA1 gene mutations, leading to a,antitrypsin (AAT) deficiency, called A(1)ATVar, which can be accessed over the World Wide Web at www.goldenhelix.org/A1ATVar. Extensive information has been extracted from the literature and converted into a searchable database, including genotype information, clinical phenotype, allelic frequencies for the commonest AAT variant alleles, methods of detection, and references. Mutation summaries are automatically displayed and user-generated queries can be formulated based on fields in the database. A separate module, linked to the FINDbase database for frequencies of inherited disorders allows the user to access allele frequency information for the three most frequent AAT alleles, namely PiM, PiS, and PiZ. The available experimental protocols to detect AAT variant alleles at the protein and DNA levels have been archived in a searchable format. A visualization tool, called VariVis, has been implemented to combine A(1)ATVar variant information with SERPINA1 sequence and annotation data. A direct data submission tool allows registered users to submit data on novel AAT variant alleles as well as experimental protocols to explore SERPINA1 genetic heterogeneity, via a password-protected interface. Database access is free of charge and there are no registration requirements for querying the data. The A(1)ATVar database is the only integrated database on the Internet offering summarized information on AAT allelic variants and could be useful not only for clinical diagnosis and research on AAT deficiency and the SERPINA1 gene, but could also serve as an example for an all-in-one solution for locus-specific database (LSDB) development and curation. Hum Mutat 30, 308-313, 2009

Stops making sense: translational trade-offs and stop codon reassignment

Background Efficient gene expression involves a trade-off between (i) premature termination of protein synthesis; and (ii) readthrough, where the ribosome fails to dissociate at the terminal stop. Sense codons that are similar in sequence to stop codons are more susceptible to nonsense mutation, and are also likely to be more susceptible to transcriptional or translational errors causing premature termination. We therefore expect this trade-off to be influenced by the number of stop codons in the genetic code. Although genetic codes are highly constrained, stop codon number appears to be their most volatile feature. Results In the human genome, codons readily mutable to stops are underrepresented in coding sequences. We construct a simple mathematical model based on the relative likelihoods of premature termination and readthrough. When readthrough occurs, the resultant protein has a tail of amino acid residues incorrectly added to the C-terminus. Our results depend strongly on the number of stop codons in the genetic code. When the code has more stop codons, premature termination is relatively more likely, particularly for longer genes. When the code has fewer stop codons, the length of the tail added by readthrough will, on average, be longer, and thus more deleterious. Comparative analysis of taxa with a range of stop codon numbers suggests that genomes whose code includes more stop codons have shorter coding sequences. Conclusions We suggest that the differing trade-offs presented by alternative genetic codes may result in differences in genome structure. More speculatively, multiple stop codons may mitigate readthrough, counteracting the disadvantage of a higher rate of nonsense mutation. This could help explain the puzzling overrepresentation of stop codons in the canonical genetic code and most variants

On the possibility of magneto-structural correlations: detailed studies of di-nickel carboxylate complexes

A series of water-bridged dinickel complexes of the general formula [Ni<sub>2</sub>(μ<sub>2</sub>-OH<sub>2</sub>)(μ2- O<sub>2</sub>C<sup>t</sup>Bu)<sub>2</sub>(O<sub>2</sub>C<sup>t</sup>Bu)2(L)(L0)] (L = HO<sub>2</sub>C<sup>t</sup>Bu, L0 = HO<sub>2</sub>C<sup>t</sup>Bu (1), pyridine (2), 3-methylpyridine (4); L = L0 = pyridine (3), 3-methylpyridine (5)) has been synthesized and structurally characterized by X-ray crystallography. The magnetic properties have been probed by magnetometry and EPR spectroscopy, and detailed measurements show that the axial zero-field splitting, D, of the nickel(ii) ions is on the same order as the isotropic exchange interaction, J, between the nickel sites. The isotropic exchange interaction can be related to the angle between the nickel centers and the bridging water molecule, while the magnitude of D can be related to the coordination sphere at the nickel sites

Comparative significance of invasive measures of microvascular injury in acute myocardial infarction

Background: The resistive reserve ratio (RRR) expresses the ratio between basal and hyperemic microvascular resistance. RRR measures the vasodilatory capacity of the microcirculation. We compared RRR, index of microcirculatory resistance (IMR), and coronary flow reserve (CFR) for predicting microvascular obstruction (MVO), myocardial hemorrhage, infarct size, and clinical outcomes, after ST-segment–elevation myocardial infarction. Methods: In the T-TIME trial (Trial of Low-Dose Adjunctive Alteplase During Primary PCI), 440 patients with acute ST-segment–elevation myocardial infarction from 11 UK hospitals were prospectively enrolled. In a subset of 144 patients, IMR, CFR, and RRR were measured post-primary percutaneous coronary intervention. MVO extent (% left ventricular mass) was determined by cardiovascular magnetic resonance imaging at 2 to 7 days. Infarct size was determined at 3 months. One-year major adverse cardiac events, heart failure hospitalizations, and all-cause death/heart failure hospitalizations were assessed. Results: In these 144 patients (mean age, 59±11 years, 80% male), median IMR was 29.5 (interquartile range: 17.0–55.0), CFR was 1.4 (1.1–2.0), and RRR was 1.7 (1.3–2.3). MVO occurred in 41% of patients. IMR>40 was multivariably associated with more MVO (coefficient, 0.53 [95% CI, 0.05–1.02]; P=0.031), myocardial hemorrhage presence (odds ratio [OR], 3.20 [95% CI, 1.25–8.24]; P=0.016), and infarct size (coefficient, 5.05 [95% CI, 0.84–9.26]; P=0.019), independently of CFR≤2.0, RRR≤1.7, myocardial perfusion grade≤1, and Thrombolysis in Myocardial Infarction frame count. RRR was multivariably associated with MVO extent (coefficient, −0.60 [95% CI, −0.97 to −0.23]; P=0.002), myocardial hemorrhage presence (OR, 0.34 [95% CI, 0.15–0.75]; P=0.008), and infarct size (coefficient, −3.41 [95% CI, −6.76 to −0.06]; P=0.046). IMR>40 was associated with heart failure hospitalization (OR, 5.34 [95% CI, 1.80–15.81] P=0.002), major adverse cardiac events (OR, 4.46 [95% CI, 1.70–11.70] P=0.002), and all-cause death/ heart failure hospitalization (OR, 4.08 [95% CI, 1.55–10.79] P=0.005). RRR was associated with heart failure hospitalization (OR, 0.44 [95% CI, 0.19–0.99] P=0.047). CFR was not associated with infarct characteristics or clinical outcomes. Conclusions: In acute ST-segment–elevationl infarction, IMR and RRR, but not CFR, were associated with MVO, myocardial hemorrhage, infarct size, and clinical outcomes

Pneumococcal carriage in sub-Saharan Africa--a systematic review.

BACKGROUND: Pneumococcal epidemiology varies geographically and few data are available from the African continent. We assess pneumococcal carriage from studies conducted in sub-Saharan Africa (sSA) before and after the pneumococcal conjugate vaccine (PCV) era. METHODS: A search for pneumococcal carriage studies published before 2012 was conducted to describe carriage in sSA. The review also describes pneumococcal serotypes and assesses the impact of vaccination on carriage in this region. RESULTS: Fifty-seven studies were included in this review with the majority (40.3%) from South Africa. There was considerable variability in the prevalence of carriage between studies (I-squared statistic = 99%). Carriage was higher in children and decreased with increasing age, 63.2% (95% CI: 55.6-70.8) in children less than 5 years, 42.6% (95% CI: 29.9-55.4) in children 5-15 years and 28.0% (95% CI: 19.0-37.0) in adults older than 15 years. There was no difference in the prevalence of carriage between males and females in 9/11 studies. Serotypes 19F, 6B, 6A, 14 and 23F were the five most common isolates. A meta-analysis of four randomized trials of PCV vaccination in children aged 9-24 months showed that carriage of vaccine type (VT) serotypes decreased with PCV vaccination; however, overall carriage remained the same because of a concomitant increase in non-vaccine type (NVT) serotypes. CONCLUSION: Pneumococcal carriage is generally high in the African continent, particularly in young children. The five most common serotypes in sSA are among the top seven serotypes that cause invasive pneumococcal disease in children globally. These serotypes are covered by the two PCVs recommended for routine childhood immunization by the WHO. The distribution of serotypes found in the nasopharynx is altered by PCV vaccination

CD1a selectively captures endogenous cellular lipids that broadly block T cell response

We optimized lipidomics methods to broadly detect endogenous lipids bound to cellular CD1a proteins. Whereas membrane phospholipids dominate in cells, CD1a preferentially captured sphingolipids, especially a C42, doubly unsaturated sphingomyelin (42:2 SM). The natural 42:2 SM but not the more common 34:1 SM blocked CD1a tetramer binding to T cells in all human subjects tested. Thus, cellular CD1a selectively captures a particular endogenous lipid that broadly blocks its binding to TCRs. Crystal structures show that the short cellular SMs stabilized a triad of surface residues to remain flush with CD1a, but the longer lipids forced the phosphocholine group to ride above the display platform to hinder TCR approach. Whereas nearly all models emphasize antigen-mediated T cell activation, we propose that the CD1a system has intrinsic autoreactivity and is negatively regulated by natural endogenous inhibitors selectively bound in its cleft. Further, the detailed chemical structures of natural blockers could guide future design of therapeutic blockers of CD1a response

Who Benefits From Teams? Comparing Workers, Supervisors, and Managers

This paper offers a political explanation for the diffusion and sustainability of team-based work systems by examining the differential outcomes of team structures for 1200 workers, supervisors, and middle managers in a large unionized telecommunications company. Regression analyses show that participation in self-managed teams is associated with significantly higher levels of perceived discretion, employment security, and satisfaction for workers and the opposite for supervisors. Middle managers who initiate team innovations report higher employment security, but otherwise are not significantly different from their counterparts who are not involved in innovations. By contrast, there are no significant outcomes for employees associated with their participation in offline problem-solving teams

Design of a multicentre randomized trial to evaluate CT colonography versus colonoscopy or barium enema for diagnosis of colonic cancer in older symptomatic patients: The SIGGAR study

Background and Aims: The standard whole-colon tests used to investigate patients with symptoms of colorectal cancer are barium enema and colonoscopy. Colonoscopy is the reference test but is technically difficult, resource intensive, and associated with adverse events, especially in the elderly. Barium enema is safer but has reduced sensitivity for cancer. CT colonography ("virtual colonoscopy") is a newer alternative that may combine high sensitivity for cancer with safety and patient acceptability. The SIGGAR trial aims to determine the diagnostic efficacy, acceptability, and economic costs associated with this new technology.Methods: The SIGGAR trial is a multi-centre randomised comparison of CT colonography versus standard investigation ( barium enema or colonoscopy), the latter determined by individual clinician preference. Diagnostic efficacy for colorectal cancer and colonic polyps measuring 1 cm or larger will be determined, as will the physical and psychological morbidity associated with each diagnostic test, the latter via questionnaires developed from qualitative interviews. The economic costs of making or excluding a diagnosis will be determined for each diagnostic test and information from the trial and other data from the literature will be used to populate models framed to summarise the health effects and costs of alternative approaches to detection of significant colonic neoplasia in patients of different ages, prior risks and preferences. This analysis will focus particularly on the frequency, clinical relevance, costs, and psychological and physical morbidity associated with detection of extracolonic lesions by CT colonography.Results: Recruitment commenced in March 2004 and at the time of writing ( July 2007) 5025 patients have been randomised. A lower than expected prevalence of end-points in the barium enema sub-trial has caused an increase in sample size. In addition to the study protocol, we describe our approach to recruitment, notably the benefits of extensive piloting, the use of a sham-randomisation procedure, which was employed to determine whether centres interested in participating were likely to be effective in practice, and the provision of funding for dedicated sessions for a research nurse at each centre to devote specifically to this trial